Method for determining the binding constant of high affinity compounds

ABSTRACT

The invention relates to a method for determining the binding constant of a compound of interest to proteins comprising the following steps: a) adding the high affinity compound to a two-chamber system, wherein the two chambers are separated by a semipermeable membrane, which is permeable for the compound of interest, and determining the amount of the high affinity compound of interest in one of the chambers after the distribution equilibrium has been reached, b) adding a sink compound to one of the chambers whereby the sink compound can not permeate the membrane, and determining the distribution coefficient of the compound of interest to the sink compound after the distribution equilibrium has been reached, c) adding an unspecific protein to the other chamber, whereby the unspecific protein can not permeate the membrane, and determining the distribution coefficient of the compound of interest to the unspecific protein in presence of a sink compound after the distribution equilibrium has been reached, and d) determining the binding constant of the test compound with the distribution coefficient of steps b) and c).

The success of a compound as drug is not only dependent of its potency,but rather of the optimal balance between the drug's strength, druginteraction, safety, pharmacokinetics, and productions costs.

Before the compound can be effective at the receptor its release,absorption, and distribution are of considerable influence. Only a freedrug (not bound) can be effective on the receptor. If the drug binds toostrong to proteins, the intensity of its pharmacological response can bereduced and it can have also an effect on the distribution volume,metabolisation, and elimination of the drug.

Is it essential to maximize the generation of an in vitro data setthrough High Throughput—ADME Screens (A=absorption, D=distribution,M=metabolism, E=excretion) to get the chemists a feedback for finding asubstance with the best physicochemical properties and to understand therelationship between chemical structure and physicochemical parameters.

A known method for determining the protein binding is e.g. anequilibrium dialysis. Start point of this method are two chambers, asample chamber and a protein chamber, which are separated by asemipermeable membrane. The MWCO of the membrane is chosen such that thetest substance is able to permeate but that the macromolecule (e.gprotein) is retained. A known concentration and volume of the testsubstance is placed in the sample chamber. Then, a known concentrationof the protein is placed in the protein chamber in a volume equivalentto that one of the substance in the sample chamber. As the testsubstance diffuses across the membrane some will bind to the protein andsome will remain free in solution. The lower the affinity of theinteraction, the higher the concentration of the test substance thatwill remain unbound at any time. The diffusion of the test substanceacross the membrane and binding of the test substance continues untilequilibrium has been reached. At equilibrium, the concentration of thetest compound free in solution is the same in both chambers. In theprotein chamber, however, the overall concentration of the testsubstance is higher due to the bound test substance (if the testcompounds binds to the protein). The concentration of free testsubstance in the sample chamber can then be used to determine thebinding characteristics of the test compound.

Another method is the use of the BIAcore technology, whereby one of thebinding partners is immobilized on a sensor chip. The other bindingpartner flows through the chip which is placed in a micro cell. Thedetection of the binding relies on the phenomenon of “surface plasmonresonance” (SPR), small changes in the reflection of monochromatic lightfrom a metallic chip that occurs when the chip's surface binds a proteinor other molecule.

However, all these methods are not suitable for determining the proteinbinding capability of high affinity substances in a HighThroughput—Assay as it is not possible to differentiate between 99% and99.99% binding with a high throughput of substances. The reason is thatthe concentration of the free test substance, which is what is analyzedin the end, is to low to be quantified.

With high affinity drugs, small changes in the amount of boundsubstances can have a profound influence on the free fraction in thebody (see table below) and thereby influence on effect and side effects.Therefore, it is of high importance to make a measurement, in this veryrange.

Example with a difference of 4% :

99% → 95% Binding  1% free → 5 % free Factor 5 80% → 76% Binding 20%free → 24 % free Factor 1.2Therefore, the present invention provides a method for determining thebinding constant of a high affinity compound of interest to proteinscomprising the following steps:

-   a) adding the high affinity compound to a two-chamber system,    wherein the two chambers are separated by a membrane which is    permeable for the compound of interest, and    -   determining the amount of the high affinity compound of interest        in one of the chambers after the distribution equilibrium has        been reached,-   b) adding a sink compound to one of the chambers (sink chamber)    whereby the sink compound can not permeate the membrane, and    -   determining the distribution coefficient of the compound of        interest to the sink compound after the distribution equilibrium        has been reached,-   c) adding an unspecific protein to the other chamber (protein    chamber), whereby the unspecific protein can not permeate the    membrane, and    -   determining the distribution coefficient of the compound of        interest to the unspecific protein in presence of a sink        compound after the distribution equilibrium has been reached,        and-   d) determining the binding constant of the test compound with the    distribution coefficient of steps b) and c).

The steps a), b), and steps c) can be performed consecutively, or theycan be performed in parallel (see FIG. 3).

Therefore, the present invention also provides a method for determiningthe binding constant of a high affinity compound of interest to proteinscomprising the following steps:

-   a) providing two-chamber systems for parallel performance wherein    the two chambers of each system are separated by a membrane which is    permeable for the compound of interest and adding the high affinity    compound to a two-chamber system, and-   determining the amount of the high affinity compound of interest in    one of the chambers after the distribution equilibrium has been    reached,-   b) adding the high affinity compound and a sink compound to one of    the chambers of a second chamber system (sink chamber) whereby the    sink compound can not permeate the membrane, and    -   determining the distribution coefficient of the compound of        interest to the sink compound after the distribution equilibrium        has been reached,-   c) adding the high affinity compound and a sink compound to one of    the chambers of a third chamber system (sink chamber) and adding an    unspecific protein to the other chamber (protein chamber), whereby    the unspecific protein can not permeate the membrane, and the    distribution coefficient of the compound of interest to the    unspecific protein in presence of a sink compound after the    distribution equilibrium has been reached, and-   d) determining the binding constant of the test compound with the    distribution coefficient of steps b) and c),-   wherein the steps a), b), and c) are performed in parallel.

Furthermore, the present application provides the use of a sink compoundfor determining the binding constant of a high affinity test compound.

A sink compound is a substance, which significantly lowers the affinityof high affinity test substances to an unspecific protein. Significantlylower the affinity means that the difference between the amount of boundtest substance to the unspecific protein in the presence and absence ofthe sink compound is statistically relevant (p≦0.05, preferably,p≦0.01). To avoid that the sink compound crosses the membrane, butallowing the test compound to pass it, the size of the sink compound ispreferably bigger by factor 2 than the size of the test compound.Preferably, a sink compound is a complexing agent. More preferably, thesink compound is Polyvinylpyrrolidone (PVP), most preferably, the sinkcompound is Polyvinylpyrrolidone K-25 (PVP 25, PVP K-25). For thepurpose of the above mentioned method a combination of sink compoundscan be used, e.g. PVP and Cyclodextrin.

The term “high affinity compound” as used herein, refers to a compoundof which under physiological conditions and in the presence of a surplusof plasma protein (e.g. Albumin) at least 95% of the total amount isbound to said plasma protein (=at most 5% of the total amount of saidcompound are in free form). Preferably, at least 97% are bound, morepreferably, at least 98% and most preferably, at least 99% are presentin bound form. Physiological conditions are defined as pH 7.0 and 37° C.In terms of the present invention, a high affinity compound or testcompound is preferably a drug.

The term “test substance” or “test compound” as used herein refers to ahigh affinity compound.

The term “binding constant” refers to constant that describes thebinding affinity between two molecules at equilibrium. It describes thestate of equilibrium between free and bound test substance.

The term “bound substance” or “bound compound” as used herein refers toa substance or compound which is bound to a protein, (in particular toan unspecific protein) or to the sink compound. The term “fractionaloccupation” refers to the fraction of test compounds that is bound. Inother words, it is the proportion between the amount of test compoundbound in equilibrium to a sink compound (c_(bound test substance)) andthe total amount of test compound (c_(total test substance)).

${{fractional}\mspace{14mu} {occupation}} = {{{fraction}\mspace{14mu} {bound}} = \frac{c_{{bound}\mspace{14mu} {test}\mspace{14mu} {substance}}}{c_{{total}\mspace{14mu} {test}\mspace{14mu} {substance}}}}$

An unspecific protein binds in contrast to a specific protein to a broadrange of compounds (unselective binding). Unspecific proteins are inparticular plasma proteins, such as e.g. serum albumin and α1 acidglycoprotein. Preferably, the unspecific protein is human serum albumin(HSA).

The membrane used in the method of the present invention is asemipermeable membrane. A semipermeable membrane is a membrane whichallows a selected species of molecules to pass through it by diffusion.Semipermeable membranes for the above mentioned assay can be passed bythe high affinity compound (and the buffer), but not by the sinkcompound or the unspecific protein. Preferably, the semipermeablemembrane is a size selective membrane.

Semipermeable membranes can be characterized by Molecular Weight Cut Off(MWCO). Preferably, MWCO is approximately half of the molecular weightof the sink compound or the unspecific protein, whichever is thesmaller. Also preferred is a proportion of 22 to 27 between themolecular weights of the test substance and the sink compound or theunspecific protein, whichever is the smaller. More preferably, theproportion is about 25. Suitable membranes for the methods of thepresent invention are commercially available, e.g. dialysis membranes.

Preferably, the concentration of unspecific protein used in the assaycorresponds to the concentration of the protein in vivo. For HSA, thephysiological concentration is around 60 mM.

The concentration of the sink compound for the optimal performance ofthe method of the invention can be determined with performing the methodof the invention with different concentrations of the sink compound(i.e. serial dilution).

The assay is performed in a two chamber system, wherein the two chambersare separated by a semipermeable membrane. The permeability of themembrane is chosen such that the test substance can pass through themembrane, but not neither the sink compound nor the unspecific protein.

The measurements of the method of the invention (steps a), b), and stepsc) and can be performed in parallel (see FIG. 3) or consecutively.Preferably, the measurements are made in parallel. In the following apreferred embodiment is described: For the reference experiment (step a)of the above described method), the compound of interest is added to oneof the chambers (see FIG. 2B) and the distribution of the compound isdetermined in this system after the distribution equilibrium has beenreached whereby the mass of the compound in at least one of the chambersis determined after distribution equilibrium has been reached.

For binding experiment 1 (step b) of the above described method), thecompound of interest and the sink compound are added to chamber in adifferent two chamber system than used for performing the referenceexperiment (see also FIG. 2B). After the equilibrium of the bindingsystem has been reached the amount of high affinity compound in thechamber without the sink compound (protein chamber) is measured and thedistribution coefficient of the compound of interest to the sinkcompound is determined.

For binding experiment 2 (step d)) of the above described method), thecompound of interest and the sink compound are added to a chamber (sinkchamber) in a different two chamber system than used for performing thereference or binding experiment (see FIG. 2C) and an unspecific proteinis added to the chamber without sink compound (protein chamber). Afteran equilibrium has reached, the amount of free test substance (not boundto the sink compound) in the sink chamber is measured and thedistribution coefficient of the compound of interest to the unspecificprotein in the presence of the sink compound is determined.

The binding constant of the high affinity compound is determined asfollows:

The binding of a test compound to an unspecific protein is a reversibleprocess which can be described by the following equilibrium (Lindup, W.E., Plasma protein binding of drugs—some basic and clinical aspects, inProgress in Drug Metabolism, L. F. C. J. W. Bridges, G. G. Gibson,Editor. 1987. p. 141-185)

[T_(u)] + [P] ⇆ [T ⋅ P]$K_{P} = \frac{\left\lbrack {T \cdot P} \right\rbrack}{\left\lbrack T_{u} \right\rbrack*\lbrack P\rbrack}$

[P]=concentrations of the unspecific protein

[T_(u)]=concentrations of test compound not bound to unspecific protein.

K_(P)=Binding constant of the test compound to the unspecific protein

[T·P]=concentration of test compound bound to nonspecific protein whenequilibrium is reached

The binding constant K_(P) can be converted to the fraction unboundf_(u) with the following formula derived from the law of mass:

$\begin{matrix}{f_{u} = {{1 - f_{b}} = \frac{100}{1 + {K_{P}*\lbrack P\rbrack}}}} & (1)\end{matrix}$

f_(b)=fraction of test compound bound to the unspecific protein

The binding constant to unspecific protein (K_(P)) (2) can be calculatedfrom the combination of partition coefficients to sink compound(DC_(sink)) (3) and to the unspecific protein in the presence of thesink compound (DC_(P′)) (3).

K _(P) =DC _(sink) *DC _(P).   (2)

From Binding Study I (with sink compound, FIG. 2B):

$\begin{matrix}\begin{matrix}{{DC}_{sink} = \frac{\left\lbrack {T - {Sink}} \right\rbrack}{\left\lbrack T_{u{({sink})}} \right\rbrack}} \\{= {\frac{m_{{Tb}{({sink})}}}{m_{u{({sink})}}}*\frac{V_{total} - V_{sink}}{V_{sink}}}} \\{= \frac{m_{T{({ref})}} - m_{{Tu}{({sink})}}}{m_{{Tu}{({sink})}}}}\end{matrix} & (3)\end{matrix}$

m_(T(ref))=Mass of test compound in equilibrium, in the absence of thesink compound and the unspecific protein (measured in one of thechambers)

m_(Tu(sink))=Mass of unbound test compound in equilibrium, in presenceof the sink compound

From Binding study II (system with sink compound and high affinitycompound, FIG. 2C):

$\begin{matrix}\begin{matrix}{{DC}_{P^{\prime}} = \frac{\left\lbrack {T - P^{\prime}} \right\rbrack}{\left\lbrack T_{u{({sink})}} \right\rbrack}} \\{= {\frac{m_{{Tb}{(P^{\prime})}}}{m_{{Tu}{(P^{\prime})}}}*\frac{V_{total} - V_{P}}{V_{P}}}} \\{= \frac{m_{T{({ref})}} - m_{{Tu}{(P^{\prime})}}}{m_{{Tu}{(P^{\prime})}}}}\end{matrix} & (4)\end{matrix}$

[T−P′]=Concentration of bound test compound to unspecific protein in thepresence of the sink compound

[T−Sink]=Concentration of test compound bound to the sink compound

m_(Tb(P′))=Mass of test compound bound to the unspecific protein inequilibrium, in the system with the sink compound and the unspecificprotein

m_(Tu(P′))=Mass of test compound not bound to the unspecific protein inequilibrium in the system with the sink compound and the unspecificprotein

V_(total) is total volume in the dialysis chamber (e.g. 200 μl)

V_(P) is the volume of unspecific protein

V_(sink) is the volume of the sink compound

Volumes can be calculated from the density and the mass of the appliedmaterial

V=mass/density (e.g. Density of human serum albumin: ρ_(HSA)=1.4 g/cm³,density of PVP25: ρ_(PVP)=1.2 g/cm3).

The amount of free and/or bound test substance can be determinedspectrophotometrically directly in a spectrophotometer or afterseparation in a HPLC system.

For all reference and binding experiments, the chamber systems arepreferably incubated under room temperature and normal pressure (e.g.overnight (about 12 hours)). For accelerating the achievement of theequilibrium the chamber can be shaken (preferably for about 1 to 3hours, more preferably for 1 to 2 hours, most preferably for about 1hour).

In a preferred embodiment, the method of the present invention isperformed in parallel in a plate comprising a plurality of a two chambersystem. These parallel performed assays be identical or they can differe.g. in the identity or the concentration of the high affinity compound.

Having now generally described this invention, the same will becomebetter understood by reference to the specific examples, which areincluded herein for purpose of illustration only and are not intended tobe limiting unless otherwise specified, in connection with the followingfigures.

FIGURES

FIG. 1 shows a dialysis chamber system consisting of 10 Teflon™ barswherein 8 of the Teflon™ bars have 12 drill holes on each site and twohave 12 drill holes on one side (FIG. 1A). Said drill holes have theform of a half cylinder (see FIG. 1B) so that two bars together form arow of 12 cylindrical wells (rows A to I). Between the Teflon™ barssemipermeable membranes (M) are inserted such that each cylindrical wellis divided into two half cylindrical chambers. The 10 Teflon™ bars withthe membranes are assembled with two stainless steel connecting rods(R).

FIG. 2 shows the dialysis chamber system during an assay. The chambercomprises two chambers (chamber 1, chamber 2) which are separated by amembrane M. The size selective membrane M separates the two chambers andis permeable for the test compound but not for the sink compound or theunspecific protein. Test compound:

; unspecific protein:

; the sink compound:

. The encircled chamber number indicates the chamber from which a sampleis taken for measurement. FIG. 2A) shows a schematic representation ofthe situation in the chamber system under reference conditions, wherebythe test compound distributes evenly in the chamber system. FIG. 2B)shows a schematic representation of the situation in the chamber systemwith the test compound and the sink compound. Test compound and sinkcompound are added to chamber 1 and influences the distribution of thetest compound as only unbound test compound can cross the membrane. FIG.2C) shows a schematic representation of the situation in the chambersystem with test compound, the sink compound, and the unspecificprotein. Test compound and sink compound are added to chamber 1 and theunspecific protein is added to chamber 2. The sink compound reduces theamount of unbound test compound available for the unspecific protein.FIG. 2D) shows a schematic representation of the situation in thechamber system with a prior art assay. A high percentage of the testcompound is bound to the unspecific protein.

FIG. 3 shows a schematic representation of the method of the presentapplication performed in parallel in 3 two-chamber systems.

FIG. 4 shows the chemical structure of beta-hydroxypropyl-cyclodextrin(A) and of a polyvinylpyrrolidon monomer (B).

FIG. 5 shows a comparison of the fractional occupation of a testcompound (Warfarin, Diclofenac) with the sink compound PVP 25 to thefractional occupation of the test compounds with a combination of sinkcompounds (beta-hydroxypropyl-cyclodextrine, PVP25). X-axis:concentration of unbound test compound in equilibrium in mM, y-axis:concentration of test compound bound.

Warfarin with PVP25;

Diclofenac with PVP25; ♦ Warfarin with PVP 25 and β-hydroxypropylcyclodextrin (

Linear); ♦ Diclofenac with PVP25 and β-hydroxypropyl cyclodextrin; (

Linear).

FIG. 6 shows a graphical representation of the fractional occupation ofa cardevilol with a combination of sink compounds(beta-hydroxypropyl-cyclodextrine, PVP25). X-axis: concentration ofunbound cardevilol in equilibrium in mM, y-axis: concentration of testcompound bound. ♦ Carvedilol PVP 25with β-hydroxypropyl Cyclodextrin

FIG. 7 shows a graphical representation of the fractional occupation ofa test compounds (Diclofenac, Warfarin, Carvedilol, Naproxen, Proxicamand Glibenclamid) with the sink compound PVP 25. x-axis: totalconcentration of cardevilol in mM, y-axis: fractional occupation of thecardevilol in % (amount of test compound bound to sink compound). ♦Diclofenac with PVP25 (

Linear); ▪ Warfarin with PVP (

Linear); ♦ Carvedilol with PVP 25 ( - - - - Linear), × Naproxen withPVP25 (

Linear); * Piroxicam with PVP25 (

Linear);  Glibenclamid with PVP25 (

Linear).

FIG. 8 shows a graphical representation of the fractional occupation ofa test compounds (Diclofenac, Warfarin, Carvedilol, Naproxen, Proxicamand Glibenclamid) with the sink compound PVP 25 in the presence of humanserum albumin (HSA). x-axis: total concentration of cardevilol in mM,y-axis: fractional occupation of the cardevilol in % (amount of testcompound bound to sink compound). ♦ Diclofenac with PVP25 (

Linear);

Warfarin with PVP (Linear); ▴ Carvedilol with PVP 25 (

Linear), × Naproxen with PVP25 (

Linear); * Piroxicam with PVP25 (

Linear);  Glibenclamid with PVP25 ( - - - Linear).

FIG. 9 shows a graphical representation of the partition ofChlorpromazine (A), Diclofenac (B), Piroxicame (C), Naproxen (D),Warfarin (E) and Glibenclamid (F) to HSA in the presence of PVP25 (▪)and to PVP25 (▴).

FIG. 10 shows a graphical representation of the determined bindingconstant with the method of the present invention compared with theliterature values.

EXAMPLES

Commercially available reagents referred to in the examples were usedaccording to manufacturer's instructions unless otherwise indicated.

Example 1 Material and Methods

1.1 Dialysis Chambers

A reusable 96-well Micro-Equilibrium dialysis Device HTD 96 chamber wasused for the following experiments. The chamber system consists of 10Teflon™ bars, wherein semipermeable membranes (see 1.2 Membranes) wereplaced between two Teflon™ bars each to form two compartment of eachwell.

The assembled Teflon™ block was inserted into a steel frame. To ensurethe sealing between the individual wells, the bars are pressed togetherwith steel plate. Said steel plate imposed counter pressure with 8 discsprings upon the Teflon™ bars.

Before each experiment, the Teflon™ bars were cleaned by rinsing fourtimes in ethanol.

1.2 Membranes

MWCO

Following MWCOs were tested in dependency of the additive:

Additive [kDa] Micellar agent 6-8; 12-14 Cyclodextrin 1 PVP 3.5PVP—Cyclodextrin-mixture 1

Membrane Material

A) HTD 96 Dialysis Membrane Strips (regenerated cellulose) with MWCO of6-8 kDa (HTDialysis, LLC, Catalog Nr 1103) and MWCO of 12-14 kDa(HTDialysis, LLC, Catalog Nr 1101).

The above mentioned membranes were hydrated with deionized water for 60minutes. Subsequently, they were treated for 20 minutes with 20% ethanoland then rinsed twice with deionized water.

B) Spektra/Por 6 (regenerated cellulose) with MWCO of 1 kDa (SpectrumLaboratories, Catalog No. 132640, 45 mmflat width/10 m length) and withMWCO of 3.5 kDa (Spectrum Laboratories, Catalog No. 132592, 45 mmflatwidth/10 m length

Removal of Heavy Metals:

Heavy Metal cleaning solution comprising EDTA (Spektrum, Cat.: 132908);1 part wash solution+9 parts H2O distilled.

The 1 h washing, then 5 min in H2O distilled.

Removal of Sulfides 0.1%:

Sulfur cleaning solution A (═Na-sulfite) and

B (=0.4% sulfuric acid) (Spektrum, Cat.: 132 906).

At 80° C. the membranes were washed with a solution comprising 2 partsH2O dist+1 part Solution A. Then the membranes were washed in 60 ° C.hot H2O for 2 min). The membranes were then washed in a solutioncomprising 100 parts water and 4 parts Solution B and then they werewashed in distilled water for some minutes.

Alternatively, pre-washed membranes were used.

C) Spektra/Por 7 (Regenerated Cellulose) MWCO 3.5 kDa (SpectrumLaboratories, Catalog No.: 132111, 45 mm flat width/5 m length

The membrane was washed in distilled water.

1.3 Sink Compounds

A) Cyclodextrine (FIG. 3)

The condition for the use of cyclodextrine is that the molecular weightis bigger than the MWCO of the used membrane to ensure that the sinkcompound is separated from the sink chamber.

The following cyclodextrins were used:

beta—Hydroxypropxyl—Cyclodextrin

gamma—Cyclodextrin.

B) Polyvinvivyrrolidon PVT' (FIG. 4)

PVP (Polyvidon, Povidon, Povidonum), Homopolymer of N-Vinylpyrrolidon

The molecular weight is usually expressed as the K value. The molecularweight is dependent of the polymerization grade (approx. 2500-3,000,000Dalton).

Experiments were done with PVP 25 (PVP K-25) with a molecular weight ofMr˜24.000, and PVP 15 (PVP K-15) with a molecular weight of Mr˜10.000.

1.4 Test Compounds

-   Warfarin ((RS)-4-hydroxy-3-(3-oxo-1-phenylbutyl)-2H-chromen-2-one),-   Furosemide(4-chloro-2-(furan-2-ylmethylamino)-5-sulfamoylbenzoic    acid)-   Diclofenac(2-(2-(2,6-dichlorophenylamino)phenyl)acetic acid)-   Verapamil(2-(3,4-dimethoxyphenyl)-5-[2-(3,4-dimethoxyphenyl)ethyl-methyl-amino]-2-(1-methylethyl)pentanenitrile)-   Chlorpromazine(3-(2-chloro-10H-phenothiazin-10-yl)-N,N-dimethyl-propan-1-amine)-   Carvedilol(3-(9H-carbazol-4-yloxy)-2-hydroxypropyl][2-(2-methoxyphenoxy)ethyl]amine)-   Naproxen((+)-(S)-2-(6-methoxynaphthalen-2-yl)propanoic acid)-   Piroxicam((8E)-8-[hydroxy-(pyridin-2-ylamino)methylidene]-9-methyl-10,10-dioxo-10λ6-thia-9-azabicyclo[4.4.0]deca-1,3,5-trien-7-one)-   Glibenclamide(5-chloro-N-(4-[N-(cyclohexylcarbamoyl)sulfamoyl]phenethyl)-2-methoxybenzamide)

1.5: Experimental Procedure

1) preparing the membranes (see example 2) and assembling the dialysischamber in example 1.

2) The protein chamber (chamber 2) is filled with buffer and the otherexcipients to the final volume of approximately 100-150 ul. It isimportant that on each side the volume is exactly the same.

Volume Per Chamber:

with pure cyclodextrines as sink compound the volume in each chamber was147 μl (γ-Cyclodextrin 2.5% (m/V))

with PVP and PVP in combination with cyclodextrines as sink compound inchamber the volume was 100 μl. (PVP 15 4% (m/V), PVP 25 4% (m/V), PVP 254% (m/V)+β-Hydroxypropyl-cyclodextrin 4% (m/V))

Buffer: 50 mM TAPSO, pH 7.4, isotonised with NaCl to the totalconcentration of 154 mM, addition excipient different depending onexcipient

End Concentration of the test compound: 0.02 mM till 0.6 mM

End concentration of HSA: 60 mM

End concentration of DMSO: 6% (V/V)

The DMSO stock solution is added at the end because it takes long butthe chambers has to be filled rather fast due to the risk that themembrane dries out.

3) All pipetting steps were automated (Tecan)

4) To prevent evaporation the Teflon™ assembly was covered with anadhesive film (Cat. Nr.: 1102; HT—Dialysis).

5) The chamber system was shaken for 1 hour, then incubated over night(approximately 12 hours). Long duration shaking should be avoided due tothe risk of higher evaporation caused by the generated heat.

6) After reaching the equilibrium, a sample from the sink chamber(reference, binding study II with sink compound and HSA), or from theprotein chamber (reference, binding study I with sink compound alone)was transferred to a half—area UV—plate and analyzed spectrometrically(spectra max,190 Plate Reader; 250-500 nm) or it was analysed by HPLC.

1.6 Analysis of Measured Data

The distribution coefficient and the binding constant can be calculatedwith binding studies.

The binding of drugs to HSA is a reversible process which can bedescribed by the following equilibrium [4]

[D _(u)]+[HSA]⇄[D·HSA]

where square brackets are used to denote concentrations and [D_(u)],[HSA] and [D·HSA] are the concentrations of non HSA-bound drug, HSA andHSA—bound drug respectively.

The binding constant K_(HSA) can be converted to the fraction unboundf_(u) with the following formula derived from the law of mass:

$\begin{matrix}{f_{u} = \frac{100}{1 + {K_{HSA} \cdot \left\lbrack {H\; S\; A} \right\rbrack}}} & (1)\end{matrix}$

The binding constant to HSA (K_(HSA)) (2) can be calculated from thecombination of partition coefficients to PVP (DC_(PVP)) (3) and to USAin the presence of PVP (DC_(HSA)) (3).

K _(HSA) =DC _(PVP) ·DC _(HSA′)  (2)

From Binding Study I:

$\begin{matrix}\begin{matrix}{{VK}_{PVP} = \frac{\left\lbrack {D - {P\; V\; P}} \right\rbrack}{\left\lbrack D_{u{({PVP})}} \right\rbrack}} \\{= {\frac{m_{D_{b{({PVP})}}}}{m_{D_{u{({PVP})}}}} \cdot \frac{V_{total} - V_{PVP}}{V_{PVP}}}} \\{= \frac{m_{D_{ref}} - m_{D_{u{({PVP})}}}}{m_{D_{u{({PVP})}}}}}\end{matrix} & (3)\end{matrix}$

From Binding study II:

$\begin{matrix}\begin{matrix}{{VK}_{{HSA}^{\prime}} = \frac{\left\lbrack {D - {H\; S\; A^{\prime}}} \right\rbrack}{\left\lbrack D_{u{({HSA}^{\prime})}} \right\rbrack}} \\{= {\frac{m_{D_{b{({HSA}^{\prime})}}}}{m_{D_{a{({HSA}^{\prime})}}}} \cdot \frac{V_{total} - V_{HSA}}{V_{HSA}}}} \\{= \frac{m_{D_{ref}} - m_{D_{u{({HSA}^{\prime})}}}}{m_{D_{u{({HSA}^{\prime})}}}}}\end{matrix} & (4)\end{matrix}$

Where [D−HSA′] and [D−PVP] are the HSA—bound drug in the presence of PVPand the PVP-bound drug respectively.

V_(total) is total volume in the dialysis chamber (200 μl) and V_(HSA)and V_(PCP) are the volumes of HSA and PVP 25, which can be calculatedfrom the density and the weighted sample

$V_{HSA} = \frac{m_{HSA}}{\rho_{HSA}}$

ρ_(HSA)=1.4 g/cm³ and ρ_(PVP)=1.2 g/cm³. For determining the density PVPhad to be melted and there are variations due to inclusion of air.Therefore, real density are based on information from BASF(manufacturer).

The above mentioned equation can be applied analogous for determiningthe concentration of the unbound/bound test compound in equilibrium withanother sink compound.

Example 2 Comparison of Beta-Hydroxypropyl-Cyclodextrine withGamma-Cyclodextrine

Membrane cut off 1 kDa, Spektra/Por 6 Concentration Warfarin 0.01 mM to0.4 mM Concentration of beta-hydroxypropyl- 1%; 5%; 10% (m/V)cyclodextrine Concentration of gamma Cyclodextrin 2.5% (m/V) Analysis UV

The results are shown in table 1. With a 10% solution ofbeta—hydroxypropyl Cyclodextrin more than 30% of the test compound canbe bound. The affinity to gamma—Cyclodextrin is considerable lower thanwith beta-hydroxypropyl-cyclodextrine (2.5 times higher concentration ofgamma—Cyclodextrin is needed to achieve the same fractional occupation).

TABLE 1 Exipient binding levels measured for Warfarin Excipient f_(b)(%)SD γ-Cyclodextrin 2.5% (m/V) 6.0 2.5 β-Hydroxypropyl-cyclodextrin 1%(m/V) 6.8 2.1 β-Hydroxypropyl-cyclodextrin 10% (m/V) 27.6 4.4

Example 3 Assay with Beta-Hydroxypropyl-Cyclodextrine as Sink Compound

The test conditions are as described above unless specificallymentioned.

-   Membrane: cut off: 1 kDa; Spektra/por 6-   Concentration test compound: 0.02 mM-0.06 mM-   Concentration of beta-hydroxypropyl-cyclodextrine: 10% (mN)-   Duration of incubation: Over night (approx. 12 h)-   Tecan-   Total volume per well or chamber: 294 μl total volume    -   (each chamber: 147 μl, see above)-   DMSO—concentration: 6% (V/V)-   Analysis: Spektra max, 50 ul

TABLE 2 Average Binding of test compounds tobeta-hydroxypropyl-cyclodextrine Test substance Carbamazepin WarfarinDiclofenac Quinine Ceftriaxone Molecular Weight 236.27 308.33 296.15324.4 554.6 Average Binding [%] 31.3 26.9 23.1 21.6 14.2

Conclusion: the lower the molecular weight of the test compound, thebetter the affinity to beta-hydroxypropyl-cyclodextrine

Example 4 Comparison of PVP 15 with PVP 25

Test conditions are described as above unless specifically mentioned.

Concentrations of the drugs: 0.02-0.6 mM

Amounts of PVP 15 and PVP25: each 40 g/l

TABLE 3 Average Binding of test compounds to PVP15 and PVP25. TestSubstance Warfarin Furosemid Diclofenac Verapamil Chlorpromazin Averagebinding [%] to PVP15 34.5 37.0 21.7 n.d. 17.0 Average binding [%] toPVP25 51.4 49.4 46.4 13.1 n.d. (n.d. = not determined)

The test substances have a higher affinity to PVP 25 than to PVP 15.Therefore, the experiments of following examples were done with PVP 25.

Example 5 Assay with Combination of PVP25 and Cyclodextrin as SinkCompound

Membrane cut off 1 kDa; Spektra/Por concentration of PVP 25 4.0 % (m/V)concentration beta-hydroxypropylcyclodextrin 4.0 % (m/V) Concentrationof the test compounds 0.02 mM to 0.6 mM (warfarin, diclofenac)

Tecan for Pipeting:

Total volume per well 100 μl per well DMSO—concentration 6% (V/V)Analysis Spektra max, 50 ul

The results are shown in FIGS. 5 and 6. The use of beta-hydroxypropylcyclodextrin and PVP25 moves the equilibrium less towards complex ofdrug and sink compound than the use of PVP 25 alone (see FIG. 6).

TABLE 4 Summary of exipient binding levels measured for Warfarin(including results from Examples 2 and 4. Excipient f_(b)(%) SDγ-Cyclodextrin 2.5% (m/V) 6.0 2.5 β-Hydroxypropyl-cyclodextrin 1% (m/V)6.8 2.1 β-Hydroxypropyl-cyclodextrin 10% (m/V) 27.6 4.4 PVP 15 4% (m/V)34.5 2.9 PVP 25 4% (m/V) 51.4 3.1 PVP 25 4% (m/V) + β-Hydroxypropyl-27.9 1.8 cyclodextrin 4% (m/V)

Example 6 Assay with PVP 25 and Human Serum Albumin (HSA)

The same test conditions were used as described in Example 5 except forthe test compound Carvedilol was used. The results are shown in FIG. 8and FIG. 9.

membrane Spektra/Por 6, MWCO 3.5 kDa, concentration PVP (sink compound)4.0% (m/V) concentration HSA (unspecific protein) 60 mM Concentration oftest compound 0.02 mM to 0.6 mM DMSO—concentration 6% volume percompartment 100 μl Control PVP alone

Tecan duration of dialysis over night (approx. 12 h) analysisHPLC/Program: ATHESA injection volume: 4 μl

HPLC Conditions:

Analytics was carried out on an HPLC—system (Agilent 1100). Theseparations were performed on a RP column (Chromolith flash, RP18e,4.6×25 mm). Samples were eluted with gradient of water with 0.05% formicacid (A) and Acetonitril (B). Gradient conditions: initial 5% B, 0.4 min95% B; lmin 95% B; 1.1 min 5% B.

Results with test compound and PVP are shown in FIGS. 7 and results withtest compounds, PVP25 and HSA are shown in FIGS. 8 and 9.

Example 7 Comparison with Literature Values

The determined binding levels of the test compound in example 6 (“newapproach”) were compared with the values determined with conventionalmethods (literature values). The results are shown in table 2 and FIG.10.

TABLE 5 binding levels of test compounds from literature and derivedfrom example 6. Compound f_(u)(%)^(newApproach) f_(u)(%)^(Literature)Lit ref. Chlorpromazine 99.44 97.80 Kratochwil, N.A., et al., Predictingplasma protein binding of drugs: a new approach. Biochem Pharmacol,2002. 64(9): p. 1355-74 Piroxicame 99.64 99.0 Dollery, C., TherapeuticDrugs. second ed. Churchill Livingstone. Vol. 2. 1999. Warfarin 99.9299.4 [Kratochwil, N.A., et al., Predicting plasma protein binding ofdrugs: a new approach. Biochem Pharmacol, 2002. 64(9): p. 1355-74Diclofenac 99.95 99.5 Kratochwil, N.A., et al., Predicting plasmaprotein binding of drugs: a new approach. Biochem Pharmacol, 2002.64(9): p. 1355-74 Naproxen 99.98 99.7 Dollery, C., Therapeutic Drugs.second ed. Churchill Livingstone. Vol. 2. 1999 Glibenclamid 99.95 99.67Inhouse (with conventional methods)

1. Method for determining the binding constant of a compound of interestto proteins comprising the following steps: a) adding the high affinitycompound to a two-chamber system, wherein the two chambers are separatedby a semipermeable membrane, which is permeable for the compound ofinterest, and determining the amount of the high affinity compound ofinterest in one of the chambers after the distribution equilibrium hasbeen reached, b) adding a sink compound to one of the chambers wherebythe sink compound can not permeate the membrane, and determining thedistribution coefficient of the compound of interest to the sinkcompound after the distribution equilibrium has been reached, c) addingan unspecific protein to the other chamber, whereby the unspecificprotein can not permeate the membrane, and determining the distributioncoefficient of the compound of interest to the unspecific protein inpresence of a sink compound after the distribution equilibrium has beenreached, and d) determining the binding constant of the test compoundwith the distribution coefficient of steps b) and c).
 2. The methodaccording to claim 1 wherein the filter is a size selective membrane. 3.The method according to claim 1, wherein the sink compound is PVP orcyclodextrine.
 4. The method according to any one of claim 2 wherein thesink compound is PVP25.
 5. The method according to any one of claims 1wherein the protein is a plasma protein.
 6. The method according toclaim 5 wherein the plasma protein is serum albumin.
 7. The methodaccording to claim 1 wherein the steps a), b) and c) are performed inparallel.
 8. The method according to claim 2 wherein the sink compoundis PVP or cyclodextrine.
 9. The method according to claim 3 wherein thesink compound is PVP25.
 10. The method according to claim 8 wherein thesink compound is PVP25.
 11. The method according to claim 2 wherein theprotein is a plasma protein.
 12. The method according to claim 2 whereinthe plasma protein is serum albumin.
 13. The method according to claim11 wherein the plasma protein is serum albumin.
 14. The method accordingto claim 2 wherein the steps a), b) and c) are performed in parallel.15. The method according to claim 3 wherein the steps a), b) and c) areperformed in parallel.
 16. The method according to claim 4 wherein thesteps a), b) and c) are performed in parallel.
 17. The method accordingto claim 11 wherein the steps a), b) and c) are performed in parallel.18. The method according to claim 17 wherein the plasma protein is serumalbumin.